Chpt 9-Catalytic Strategies Flashcards
Four Classes of Enzymes
1) Serine Protease
- Ex: Chymotrypsin
2) Carbonic Anhydrase
- Ex: Carbonic Anhydrase
3) Restriction endonuclease
- Ex: EcoR V
4) Nucleoside monophosphate (NMP) kinases
- Ex: NMP kinase
Basic Principles to stabilize Transition States
BE CABAM
1) Binding Energies
2) Covalent Catalysis
- Active site contains a reactive group (nucleophile) that becomes covalently attached to the substrate during the reaction
3) General Acid Base Catalysis
- a molecule serves as a proton donor or acceptor during the reaction
- Ex: histidine
4) Catalysis by Approximation
- Bringing two substrates near to each other
5) metal ion catalysis
- metals may serve as nucleophiles or electrophiles
- metals may also increase enzymes affinity for substrate
Proteases Def
-Cleave peptide bonds (proteins) by hydrolysis (addition of water)
Common features of proteases
- activate water or another nucleophile
- polarize the peptide carbonyl group
- stabilize tetrahedral intermediate
Types of proteases
- Serine Proteases
- Cysteine proteases
- Aspartyl Proteases
- Metalloproteases active site
Chymotrypsin
- Function
- Mech
- Synthesize/activated
- Structure
- binding pocket
Serine Protease
- hydrolyzes proteins in small intestine
- cleaves peptide bonds on the carboxyl side of aromatic (W, Y, F) or large Hydrophobic (M) amino groups
- Catalytic Triad-serine, histidine and Aspartic acid
2 step mechanism
1) Acylation of Enzyme
2) deacylation of enzyme
Synthesized as 245 amino acid Zymogene (precursor) in pancreas (acinar cells)
-chymotrypsinogen
Activated by proteolytic cleavage in cascade
3 subunits held together by 4 disulfide bridges
-2 intrachain and 2 interchain
Binding pocket contains large hydrophobic aa
In chymotrypsin how do we identify catalytically active serine
Chymotrypsin contains 28 serines:
-only one reacts with DIPF-Diisopropylphosphofluoridate
Chymotrypsin: Chromogenic Substrate
Artificial chromogenic substrate
-N-acetyl-L-phenylalanine p-nitrophenyl ester
Produces Yellow Product
-p-nitrophenolate
Substrate considers ester bond instead of amide bond found in proteins
Chymotrypsin Kinetics
Obeys Michaelis Menten Kinetics
Rxn monitored by Stop flow method
- reaction appears to occur in two phases (steps)
1) Rapid Burst
2) Slower steady state reaction
Serine Proteases: Binding Pocket
Adds specificity (each contains catalytic triad)
1) Homologues of Chymotrypsin
- cleaves peptide bonds after aromatic/long hydrophobic amino acids
- pocket lined with hydrophobic residues (W, G, M)
2) Trypsin
- cleaves peptide bond after long, positively charged side chains (R, K)
- Binding pocket contains Asp
3) Elastase
- cleaves peptide bonds after small chains (A, S)
- Binding pocket contains Val
Other Enzymes use Catalytic Triad
Nonhomologues of chymotrypsin that use catalytic triad:
1)Carboxypeptidase II (homologues to esterases) -Wheat -lack 1', 2', and 3' similarity to chymotrypsin -uses catlytic triad
2)Subtilisin (from bacillus amyloliquefaciens) -archaea, bacteria, eukarya -lacks 1', 2', and 3' similar to chymotrypsin -uses a catalytic triad
Oxyanion Hole
Stabilizes the tetrahedral intermediate of carbonyl carbon by stabilizes the negative charge of oxygen
-Hydrogen bond with N-H of peptide backbone
Cysteine Proteases Active Site
Cysteine-Nucleophile
Histidine-activates cysteine
Ex: Papain and Cathepsins
Aspartyl Proteases Active Site
Two Aspartic Acids activate water molecule creating a nucleophile
Ex:
- Renin- regulates BP
- Pepsin-digestive enzyme
Metalloproteases Active Site
Bound Metal (Zinc) activates water molecule creating a nucleophile
Ex: Thermolysin and Carboxypeptidase A
